Incubation of (3Z)-nonenal (NON) with the 269,000-g particle fraction of seed homogenate of the broad bean (Vicia faba L.) afforded (2E)-4-hydroxy-2-nonenal (HNE) as the principal product. One pathway of HNE formation consisted of initial oxygenation of NON into (2E)-4-hydroperoxy-2-nonenal (HPNE) by a novel (3Z)-alkenal oxygenase activity, followed by conversion of HPNE into HNE by a previously recognized hydroperoxide-dependent epoxygenase. The hydroperoxide intermediate was detected in coincubations of NON and oleic acid, in which experiments the HPNE generated from NON supported epoxygenase-catalyzed epoxidation of oleic acid into 9,10-epoxystearic acid. Furthermore, by using an enzyme preparation in which the epoxygenase had been inactivated by pretreatment with hydrogen peroxide it was possible to isolate and characterize racemic (4R,4S) HPNE following incubation of NON. Although the (3Z)-alkenal oxygenase resembled a lipoxygenase in its action, it was not inhibited by the lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In a second pathway, HNE was produced by rearrangement of 3,4-epoxynonenal, which was in turn formed from NON by a reaction catalyzed by hydroperoxide-dependent epoxygenase. Support for this pathway came from experiments in which 18O-labeled HNE was isolated following coincubation of NON and 13-18O-labeled linoleic acid 13-hydroperoxide. The existence of 3,4-epoxynonenal as a transient intermediate in HNE biosynthesis was further demonstrated by the isolation of 3,4-epoxynonenal (61% (4R)-configuration) as a trapping product in short time incubations interrupted by addition of sodium borohydride. The two pathways established for biosynthesis of HNE involved the hydroperoxide-reducing and the olefin-epoxidizing activities of hydroperoxide-dependent epoxygenase. In the absence of extraneous olefins and hydroperoxides the two pathways would be tightly coupled and follow the stoichiometry: 2NON + 1O2-->2HNE. It was also shown that the V. faba particle fraction catalyzed oxygenation of (3Z)-hexenal into (2E)-4-hydroxy-2-hexenal.